The potential importance of CD1d-restricted V124-invariant Natural Killer T cells (NKTs) for antitumor immunity and immunotherapy has been demonstrated in multiple models of cancer as well as in recent oncology clinical trials. Recent findings by our own and other labs suggest that while NKTs lack direct effect on tumor cells in the majority of solid tumors, they mediate antitumor immune responses via specific interactions with CD1d- positive subsets of myelomonocytic cells. We have recently demonstrated that one way by which NKTs may control tumor growth is by specific recognition and killing of tumor-associated macrophages (TAMs), thereby removing essential IL-6-STAT3-mediated growth support from tumor cells. Our preliminary data suggests that there is a stepwise CCL2- and CCL20-mediated NKT-cell localization to the tumor site, in which NKTs interact with CD1d+ TAMs and immature dendritic cells (iDCs) in the context of the tumor microenvironment. We have found that monocytic cells strongly up-regulate CCL20 expression in response to hypoxia-mediated signal(s) from neuroblastoma (NB) cells, leading us to hypothesize that tumor hypoxia regulates NKT cell localization and innate antitumor activity in NB. Because CCL20 is also a potent chemoattractant for iDCs, NKT cell interaction with CD1d+ iDCs at the tumor site may lead to the generation of protective adaptive immune response. The following specific aims will test these hypotheses: 1) to examine the mechanism by which tumor hypoxia regulates NKT cell localization and innate anti-tumor activity in NB; 2) to examine the role of NKT cell interaction with CD1d+ myeloid cells at the tumor site in the mechanism of adaptive anti-tumor immune response against NB. Using established in our lab humanized NB/TAM model and human ex vivo expanded NKTs, we will evaluate the role of hypoxic signaling in CCL20-dependent NKT-cell co-localization with TAMs and the role of NKT cell interaction with TAMs in the mechanism of innate NKT cell-mediated antitumor activity. Then, using NB9464-OVA murine NB model, we will study how NKT-cell interaction with CD1d+ myeloid cells at the tumor site may lead to activation and maturation of iDCs, followed by improved presentation of tumor-derived antigens to CD8 T cells and generation of protective adaptive immunity. The results of the proposed investigation are expected to reveal novel mechanisms that govern NKT-cell localization to the tumor site and their function in the antitumor immunity.